With a view to breeding agriculturally advantageous new plant varieties, cross breeding in which two plants are crossed and progeny is selected, mutation breeding in which mutation is induced in a plant, and other methods have conventionally been practiced. Recently, with the progress of biotechnology, genetically modified plants have been bred by introducing a useful gene and causing its function to be expressed.
Breeding a New Variety by Introducing an Individual Gene
In order to breed a new variety by genetic engineering, it is usually required at a first step to isolate a gene and analyze its function. Recent years have seen a dramatic increase in molecular biological findings about plant genes and the genomic DNA sequences of many species have been determined with many partial—as well as full-length cDNA clones being isolated and determined for their sequences. However, many of the heretofore cloned putative gene functions are simply based on the information that the nucleotide sequences of genetic coding regions or the amino acid sequences deduced therefrom of the proteins are similar to the sequences of already discovered enzyme genes and the like and in order to verify the function of a particular gene, one must make sure that the expression of the gene and its phenotype agree in a transformant. As a result, considerable time and labor is required to unravel the functions of individual genes and little progress has been made in this aspect. An attempt is being made to verify the functions of a gene by isolating a full-length cDNA clone, linking it to a suitable promoter and terminator and transforming it. As an improved version of this attempt, a technique has been developed that comprises introducing a library of full-length cDNAs into a plant and making an exhaustive analysis of functions of the genes (WO 03/018808A). However, in those approaches, the promoter is not what is inherently associated with the gene and introns as well as other gene expression regulating functions have been removed, so it is not expected for the genes to be expressed to exhibit the inherent functions. As a further problem, splicing of some genes is shown to be alternative (Jordan et al. Trends in Plant Sciences 7:392-398, 2002), so the cDNA clones obtained may have lost their inherent functions. As a matter of fact, the phenotypic variations observed in such transgenic plants do not have much utility for the purpose of breeding a new variety.
In recent years, techniques in bioinformatics are employed to deduce coding region that is translated into the protein, and promoter, intron and other regions of a gene. Modes of gene expression are investigated by the microarray technology using DNA fragments. A number of function-deficient variants have been prepared by the gene knockout technique and are used in function analysis of genes. In addition, transformants having enhanced gene expression are prepared by activation tagging and used in function analysis of genes. To unravel the interrelationship between proteins encoded by genes, two-hybrid system is employed.
In the deduction of gene function by bioinformatics, the finding obtained from the relation between the function and structure of a known protein and the sequence of the gene encoding it are often employed to search for the yet to be known function of a gene. However, recent studies have shown that there are many cDNAs that are not translated into proteins, or which permit transcription of a mRNA-like RNA but not produce a protein. There are also many genes that function as low-molecular weight RNAs after transcription. Therefore, in the bioinformatics techniques proposed to date, there are many genes on genomic DNA that present difficulty in unraveling their functions. Therefore, deducing gene functions is not easy even if such latest techniques are fully exploited.
As noted above, analysis of gene functions is not easy even today. And even if a gene function is specified, it is difficult for the above-mentioned methods involving the transformation of individual genes to breed a new variety that is improved in traits whose expression will be improved in so-called heterosis or in quantitative traits.
In order to capture a gene in a certain organism that brings about a known phenotype possessed by said organism, an attempt is being widely made that comprises constructing genomic libraries from said organism, introducing the libraries with a plasmid into a microorganism such as yeast or bacterium to prepare transformed cells, selecting a particular transformed cell on the basis of the information known for said known phenotype, for example, information such as the transcript of said gene, and employing the selected transformant to clone the desired-gene (shotgun cloning) (Dairi et al. Mol Gen Genet 262:957-964, 2000).
In one application of shotgun cloning, a plant genomic library was transformed by introducing it into a plant, rather than a microorganism (Klee et al. Mol Gen Genet 210:282-287, 1987). In this experiment, a genomic library was constructed from an Arabidopsis transformant prepared by introducing a kanamycin resistance gene from a microorganism. Petunia leaf discs were infected with mixed strains of Agrobacterium containing the genomic clones in order to select kanamycin resistant petunia cells, namely, petunia cells harboring the kanamycin resistance gene derived from the Arabidopsis transformant. As a result, it was shown that the microorganism derived kanamycin resistance gene in the Arabidopsis genome could be captured after introduction into petunia by transformation.
Further disclosed in connection with Arabidopsis was a case in which a genomic library was constructed from a mutant showing chlorosulfuron resistance due to mutation in the acetohydroxy acid synthase (AHAS) gene and three genomic clones harboring the mutant AHAS gene were isolated and introduced into tobacco, producing chlorosulfuron resistant transformants (Olszewski et al. Nucleic Acid Res. 16:10765-10782, 1988).
These studies disclose techniques in which genomic libraries are used to transform plant cells and the gene cloning is performed. However, they have not succeeded in capturing any unknown gene of the donor plants of the genomic libraries, nor in improving the plants by the introduction of the unknown genes. Given those techniques, it is still difficult to breed a new variety that is improved in agriculturally useful traits, particularly in traits whose expression will be improved in so-called heterosis or in quantitative traits.
Heterosis
Heterosis is a phenomenon in which the F1 generation of a cross between inbred lines is superior to the parental lines. In heterosis, various trait improvements are recognized, such as higher vigor of the entire plant, larger plant and organs, higher yield, rapid growth, greater resistance to diseases and pests, greater resistance to various environmental stresses including drought, high temperature and cold temperature, increase or decrease in a specified component, and increase or decrease in a specified enzyme activity, and many of these traits are extremely advantageous in agriculture. A heterosis based breeding method that has been employed from old times in order to improve cultivated plants is F1 hybrid breeding in which different parents are crossed to create a new variety and this has made great contribution to breeding superior varieties of many crops including maize. However, F1 hybrid breeding requires a large number of steps such as development and improvement of the breeding population, the development of inbred lines, examination of general combining ability, examination of specific combining ability, and the selection of F1 variety. In addition, each of these steps requires a lot of time and labor. What is more, while heterosis often produces great efficacy in the crossing of genetically distant parents, in the case where the relation between the parents is remote, crossing often does not produce fertility, thus limiting the range of species that can be crossed.
The molecular mechanism for heterosis is yet to be unraveled. Even the latest textbook on thremmatology states as follows: “the causal factors (in heterosis) at the physiological, biochemical, and molecular levels are today almost as obscure as they were at the time of the conference on heterosis held in 1952” (Genetics and Exploitation of Heterosis in Crops, p. 173, ed. Coors and Pandey, 1999, American Society of Agronomy, Inc. and Crop Science Society of America, Inc., Madison, Wis., U.S.A.)
Interesting reports on heterosis in maize were recently made. They are Fu and Dooner, Proc. Natl. Acad Sci USA 99:9573-9578, 2002 and Song and Messing, Proc Natl Acad Sci USA 100:9055-9060, 2003. In both reports, the authors investigated intervarietal differences in nucleotide sequence noting specific loci in maize, and consequently showed that the intervarietal differences were considerably greater than in self-fertilizing crops such as rice.
These findings are interesting because they show that in cross-fertilizing crops such as maize which tend to develop heterosis, the sequences of genomic DNA have greater intervarietal differences than in self-fertilizing crops; yet, it cannot be said they have reasonably unraveled the molecular mechanism for heterosis.
Thus, no insight has yet been gained into the mechanism for heterosis at the molecular level. However, at the level of classical genetics, it has been suggested that the following various genetic interactions are involved in heterosis.
A) Dominance Effect
Traits for which heterosis is observed are governed by a large number of loci in various linkage groups, and in each locus, an allele advantageous for survival and productivity is often considered to be dominant whereas a disadvantageous allele is recessive. Since there are many loci in linkage, it is almost impossible to obtain a plant line in which advantageous alleles are homozygous for all of such loci. However, F1 plants can possess all the advantageous alleles from the parents so that heterosis is induced.
B) Over-Dominance Effect
In a large number of loci, the case where two alleles are heterozygous is sometimes more advantageous in survival and productivity than the case where the locus is homozygous, and the sum of such effects brings about heterosis.
If over-dominance effect exists in a locus having particularly great effect, one can observe heterosis due to the over-dominance effect of that single locus. This phenomenon is called single-gene heterosis or single-locus heterosis. Although not contributing to any particular phenotype in the original plant, this is a gene or locus that brings about a useful phenotypic variation by the interaction between genes in another plant. Known examples of genes or loci that exhibit such property are the alcohol dehydrogenase gene in maize (Schwartz, Theor Appl Genet 43:117-120, 1973) and the purple plant locus in maize (Hollick and Chandler, Genetics 150:891-897, 1998).
C) Interaction of Non-Allelic Genes
Traits advantageous for survival and productivity are sometimes brought about in F1 hybrids as synergism between different genes. The sum of the effects of a large number of genes exhibiting such property brings about heterosis. The interaction between non-alleles is also called epistasis.
D) Interaction Between Nuclear Genes and Cytoplasmic Genes
Through the interaction between nuclear genes and cytoplasmic genes, traits advantageous for survival and productivity are sometimes expressed in F1 hybrids.
The various types of interaction between multiple genes is considered to induce heterosis. Stuber (Plant Breeding Reviews 12:227-251, 1994) reviews a large number of references that show examples of the involvement of those types of interaction of genes and emphasizes that heterosis is governed by a large number of genetic factors. Li and Yuan (Plant Breeding Reviews 17:15-158, 2000) also consider that heterosis is caused by the combination of the above-mentioned various effects.
Thus, heterosis is governed by a large number of genetic factors, so it has been difficult for the prior art technology to breed a new variety that is further improved in traits whose expression is known to be higher in heterosis.
Quantitative Traits
Traits that can be improved in expression by heterosis are often “quantitative traits”, and it is not easy to genetically analyze quantitative trait loci (QTL) which govern heterosis. Nevertheless, with the recent advances in molecular biological techniques, it has become possible to perform genetic analysis of QTL using DNA markers. As a matter of fact, there are cases for successful identification of chromosomal sites containing QTL that govern certain quantitative traits. In addition, studies are being made to clone agriculturally useful genes by molecular biological techniques using genetic maps.
In some organisms, many molecular markers have been identified on chromosomes to help construct genetic maps based on the linkage analysis of the markers. Their physical relative positions have also become clear by linking long cloned genomic DNAs.
In organisms for which genetic maps have been constructed, attempts to unravel the physical positions of genes that govern those traits and isolate such genes have been made by linkage analysis of traits that exhibit specified phenotypes and their markers, and subsequent chromosome walking. As a matter of fact, several genes have been isolated by this technique (map-based cloning).
However, in standard QTL analysis, a QTL-containing site can only be identified in an approximate manner and only DNA fragments theoretically harboring a large number of genes can be identified as QTL-containing DNA fragments. It is not easy to identify such fragments as those capable of being cloned or as those that can be introduced into a plant by transformation. In addition, the task of constructing a detailed genetic map, specifying a gene of interest on the basis of the map information and cloning the gene requires a considerable amount of time and labor. In fact, there are only few cases in which DNA fragments that could increase quantitative traits were cloned on the basis of QTL analysis.
Constructing Genomic DNA Libraries and the Technology of Transformation with Genomic Fragments
The technology of constructing libraries of plant genomic fragments is known. Using transformation vectors that can be used to transform plants in the process is also known. For example, vectors are known that can be used for cloning large (40-80 kb) DNA fragments and which permit gene transfer into plants (Liu et al. Proc. Natl. Acad. Sci. USA 96:6535-6540, 1999). Experimental attempts have also been made to introduce plant genomic fragments as individual clones into higher plants. However, no one has ever made an attempt in such a way that a large number of genomic fragments that constitute a genomic DNA library are individually introduced into plants when the functions of these fragments are unknown.
It is also known that the use of a genomic clone sometimes results in a higher gene expression than when the corresponding cDNA clone is used. As a matter of fact, when a genomic fragment harboring a certain gene (maize phosphoenolpyruvate carboxylase) was introduced into a plant (rice), an extremely high-level expression of the foreign gene was observed (Ku et al. Nature Biotechnol. 17:76-80, 1999). Other reports relate to experiments in which three 40-80 kb genomic clones from Arabidopsis were individually transferred back into Arabidopsis (Liu et al. Proc Natl Acad Sci US 96:6535-6540, 1999; Shibata and Liu Trends in Plant Sci 5:354-357, 2000). Two of the clones were introduced into an Arabidopsis line that had lost gravitropism due to mutation at the locus contained in those clones and the recovery of the normal gravitropic response was confirmed.
The above findings suggest that genes in organisms, in particular, genes in multi-cell organisms are controlled with regard to their expression level in a complex way by temporal and spatial distributions of the genes in the organisms and environmental conditions such as external stimuli; in other words, the importance of a particular gene is determined by the time and extent of its expression as well as by the tissue and cell in which it is expressed, and the timing of its expression. Thus, in order to unravel gene functions including these sophisticated gene regulations, the promoter, intron, enhancer, structural gene, splicing site and all other extensive gene expression regulating factors that are contained in the genomic fragment of the particular gene must be clarified. However, this task requires a considerable amount of labor and time, making it difficult to unravel the interaction between many genetic factors
Patent Document
                1: WO 03/018808 ANon-Patent Document            1: Jordan et al. Trends in Plant Sciences 17:392-398, 2002    2: Dairi et al. Mol Gen Genet 262: 957-964, 2000    3: Klee et al. Mol Gen Genet 210:282-287, 1987    4: Olszewski et al. Nucleic Acid Res. 16:10765-10782, 1988    5: Genetics and Exploitation of Heterosis in Crops, p. 173, ed. Coors and Pandey, 1999, American Society of Agronomy, Inc. and Crop Science Society of America, Inc., Madison, Wis., U.S.A.    6: Fu and Dooner, Proc. Natl. Acad Sci USA 99:9573-9578, 2002    7: Song and Messing, Proc Natl Acad Sci USA 100:9055-9060, 2003    8: Schwartz, Theor Appl Genet. 43:117-120, 1973    9: Hollick and Chandler, Genetics 150:891-897, 1998    10: Stuber, Plant Breeding Reviews 12:227-251, 1994    11: Li and Yuan, Plant Breeding Reviews 17:15-158, 2000    12: Liu et al. Proc Natl Acad Sci USA 96:6535-6540, 1999    13: Ku et al. Nature Biotechnol. 17:76-80, 1999    14: Shibata and Liu Trends in Plant Sci 5:354-357, 2000